A panel of genotypically and phenotypically diverse clinical Acinetobacter baumannii strains for novel antibiotic development

ABSTRACT Acinetobacter baumannii is one of the most important pathogens worldwide. The intrinsic and acquired resistance of A. baumannii, coupled with the slow pace of novel antimicrobial drug development, poses an unprecedented and enormous challenge to clinical anti-infective therapy of A. baumannii. Recent studies in the field of pathogenicity, antibiotic resistance, and biofilms of A. baumannii have focused on the model strains, including ATCC 17978, ATCC 19606, and AB5075. However, these model strains represent only a limited portion of the heterogeneity in A. baumannii. Furthermore, variants of these model strains have emerged that show significant diversity not only at the genotypic level but also reflected in differences at the phenotypic levels of capsule, virulence, pathogenicity, and antibiotic resistance. Research on A. baumannii, a key pathogen, would benefit from a standardized approach, which characterizes heterogeneous strains in order to facilitate rapid diagnosis, discovery of new therapeutic targets, and efficacy assessment. Our study provides and describes a standardized, genomically and phenotypically heterogeneous panel of 45 different A. baumannii strains for the research community. In addition, we performed comparative analyses of several phenotypes of this panel. We found that the sequence type 2 (ST2) group showed significantly higher rates of resistance, lower fitness cost for adaptation, and yet less biofilm formation. The Macrocolony type E (MTE, flat center and wavy edge phenotype reported in the literature) group showed a less clear correlation of resistance rates and growth rate, but was observed to produce more biofilms. Our study sheds light on the complex interplay of resistance fitness and biofilm formation within distinct strains, offering insights crucial for combating A. baumannii infection. IMPORTANCE Acinetobacter baumannii is globally notorious, and in an effort to combat the spread of such pathogens, several emerging candidate therapies have already surfaced. However, the strains used to test these therapies vary across studies (the sources and numbers of test strains are varied and often very large, with little heterogeneity). The variation complicates the studies. Furthermore, the limited standardized resources of A. baumannii strains have greatly restricted the research on the physiology, pathogenicity, and antibiotic resistance. Therefore, it is crucial for the research community to acquire a standardized and heterogeneous panel of A. baumannii. Our study meticulously selected 45 diverse A. baumannii strains from a total of 2,197 clinical isolates collected from 64 different hospitals across 27 provinces in China, providing a scientific reference for the research community. This assistance will significantly facilitate scientific exchange in academic research.

A cinetobacter baumannii is highly viable pathogen that is widely distributed not only in the environment but also in the hospital, where it is an important causative agent of hospital-acquired infections. A. baumannii belongs to the notorious ESKAPE (Entero coccus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomo nas aeruginosa, and Enterobacter spp.) group of microorganisms.High morbidity and mortality rates due to this group of bacteria place a great burden on healthcare systems worldwide (1).It is estimated that 45,900 people in the United States are infected with A. baumannii each year (2), and the mortality rate from A. baumannii infections in intensive care units can be as high as 54% (3).
Carbapenems are often used as the first-line drugs in the treatment of multidrug resistance (MDR) to A. baumannii; however, in recent years, carbapenem-resistant A. baumannii (CRAB) have been frequently reported, with the overall resistance rates showing a worrying increase (4).In 2018, the World Health Organization (WHO) evaluated 25 resistance patterns in 20 bacterial species based on 10 criteria such as mortality, healthcare burden, community burden, resistance prevalence, and 10-year resistance trends.The WHO ranked CRAB to be of critical priority, highlighting the urgent need for increased research and approaches that allow us to understand the pathogen and tackle infections in the clinic (5).The dissemination of CRAB is predominantly driven by a few dominant clones, including the ST2, ST1, ST79, and ST25 lineages, with ST2 predominantly present on a global scale (6).The main mechanism of carbapenem resistance in A. baumannii is the production of class D b-lactamases.bla OXA-23 , bla OXA-58 , and bla OXA-24 are the most common acquired carbapenemases in A. baumannii, and they vary between countries and regions, but are mainly dominated by bla OXA-23 , which is located on chromosomes as well as on plasmids (7,8).bla OXA-23 is mediated by Tn2006, Tn2007, Tn2008, Tn2008B, and Tn2009 for translocation in CRAB, and there are geographical differences in the distribution of these transposons (9,10).
The genome of A. baumannii is extremely plastic, with a conserved core genome estimated to account for only 14.76% (2,009/13,611) of the pan-genome, which is decreasing with the number of strains sequenced, indicating the extensive heteroge neity of A. baumannii strains (11). A. baumannii can acquire or maintain antibiotic resistance through insertion sequences, integrons, transposons, prophages, and other mobile elements (12).In addition, the natural transformation ability of A. baumannii contributes to its rapid acquisition of antibiotic resistance genes, virulence factors, and adhesion-associated factors, allowing the host to acquire new functions, that is, phenotypic heterogeneity (13,14).
A. baumannii has a diverse population, with at least 237 capsular locus types and 22 lipooligosaccharide outer core types (15,16).Current studies in the field of pathogenic ity, antibiotic resistance, and biofilm of A. baumannii have focused on model strains such as ATCC 17978, ATCC 19606, and AB5075.However, these model strains represent only offer a glimpse into the heterogeneity of A. baumannii.Furthermore, variants of these model strains have emerged that show significant diversity not only at the genotypic level but also reflected in differences at the phenotypic levels of capsule, virulence, pathogenicity, and antibiotic resistance (17)(18)(19).More surprisingly, an ISAba13 insertion resulted in phenotypic changes that were distinct from the parental strain, and such insertional sequence transfer events are widespread (18).Therefore, continued use of these three limited strains is clearly inappropriate.We propose to make a collection of bacteria available to the community, which represents the wide heterogeneity of genotypes and phenotypes, to use this subset as a reference not only for scientific research but also for the rational treatment of A. baumannii infections.
In this study, we established a reference panel of 45 clinical isolates of A. baumannii based on ST Oxford typing analysis and described and evaluated their genotypic and phenotypic differences.Furthermore, we examined the strains within the panel for variations in genetic backgrounds and phenotypic traits, encompassing macrocolony morphology, resistance rate, biofilm formation, and growth rate.We hope that this panel collection reflects the wide diversity of A. baumannii strains and provides the scientific community with a scientific reference for development of novel drugs and therapeutic regimens against this species.At the same time, it can also serve as a database and collection to be used by researchers to quickly and rationally select model strains based on specific biological questions.

Collection of a panel of A. baumannii strains
All A. baumannii strains were obtained from clinical specimens collected from 64 different hospitals in China between January 2009 and December 2010.A total of 2,197 non-duplicate A. baumannii strains were included, and they were widely distributed in 27 different provinces and regions, as described previously (20).Based on the diversity of the Oxford multilocus sequence typing (MLST) scheme, we ultimately selected 45 non-duplicate A. baumannii isolates representing the diversity of the collected species.

Antimicrobial susceptibility testing (AST)
The minimal inhibitory concentrations (MICs) of cefepime, ceftazidime, imipenem, meropenem, ciprofloxacin, amikacin, gentamicin, colistin, eravacycline, and tigecycline were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (30).Escherichia coli ATCC 25922 was used as the quality control strain in antimicrobial susceptibility testing.The breakpoints of tigecycline were interpreted following the guidelines of Food and Drug Administration (FDA) for Enterobacterales.The breakpoints for eravacycline were interpreted according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for Enterobacterales, and the other antibiotics were interpreted according to the breakpoints recommended by (30), M100-S32) (30).

Macrocolony morphology
Macrocolony morphology assays were performed as described previously (11) .Bacterial single colonies were selected and incubated overnight at 37°C at 200 rpm in Muel ler-Hinton Broth (MHB).Five microliters of overnight bacterial suspension was added onto Columbia Agar containing 5% sheep blood (Becton, Dickinson and Company, Franklin Lakes, NJ).Plates were incubated non-inverted at room temperature for 6 days, followed by photographic recording with a camera.The experiment was biologically repeated three times to confirm the consistency of macrocolony morphology.Subse quently, a representative picture of each group of bacteria was selected and shown in Supplementary Material 1.

Virulence in the Galleria mellonella infection model
The G. mellonella infection model was established as described previously (31).Briefly, overnight cultures of bacteria were washed with PBS and diluted to approximately 1 × 10 7 CFU/mL.G. mellonella larvae were injected with 10 µL of the bacterial suspension into the first left prolegs (10 per group).Larvae were injected with 10 µL PBS as a combined trauma and solvent control.Incubation was carried out at 37°C in the dark.The experiment was repeated three times per group for 7 days.The number of surviving larvae was counted daily, and the log-rank test was performed using GraphPad Prism 8.4.3.

Growth rate
Growth rates were determined as described previously (32).Three independent cultures of A. baumannii isolates were grown overnight and diluted to 1:100 in MHB, and then a 200-µL aliquot was placed into a flat-bottomed 100-well plate (triplicate experiments).The plates were incubated at 37°C.The optical density at 600 nm of each culture was measured every 5 minutes for 20 hours using a Bioscreen C MBR machine (Oy Growth Curves Ab Ltd., Finland).Growth rates were estimated from OD 600 curves using an R script.ATCC17978 was used as a control for comparison with each isolate.

Biofilm formation ability
Biofilm formation assays were performed as previously described, with minor modifications (33).Overnight cultures were diluted to 1:100 and transferred to 96-well cell culture dishes at 200 µL per well and incubated at 37°C for 24 hours.Each culture was added to three wells.The wells were washed three times with phosphate buffer solution (PBS) to remove unattached bacteria.The cells were stained with 0.1% crystal violet for 15 minutes and then washed with PBS to remove excess dye.Then, 95% ethanol was added and gently shaken for 20 minutes to release the dye, and the absorbance at OD 550 was measured.Three independent experiments were carried out.

Statistical analysis
Fisher's exact test was used to compare the categorical variables between the two groups, and independent sample t-test was used to compare the differences between the sample means of the two groups.SPSS 21.0 software was used for data analysis, and P < 0.05 denotes statistically significant differences..

Diversity of the A. baumannii panel
To maximize genetic diversity, 45 strains were selected as representatives of the large microbial diversity of A. baumannii based on ST Oxford typing.The strains comprising this panel were widely distributed, covering 19 provinces in China (Fig. 1A).By analyzing the genotypic and phenotypic differences of these 45 strains in terms of the antibiotic resistance profile, capsule type, and virulence phenotype, we obtained insights of the characteristics of the strains.We constructed a core phylogenetic tree of the strains in the final panel and those from the NCBI database representing the main international clones (ICs) prevalent in hospitals worldwide (IC1-IC11), with a total of 223 genomes included in this analysis.The phylogenetic tree showed that the strains in the panel formed seven groups: four clusters and three singletons.Cluster 1 and cluster 5 are closely related to IC2 and IC8, respectively, while singleton 3 clusters together with IC1 (Fig. 1B).In addition, there were eight strains that did not belong to any of the ICs depicted in this analysis.These strains are related to ICs, whereas they are too heteroge neous to be included in any major human ICs.Subsequently, we further analyzed the 45 A. baumannii strains based on single-nucleotide polymorphisms (SNPs) in the core genome, with a maximum difference of 58,373 SNPs.With the exception of individual strains, such as XH1024 with XH1029 and XH1031 from the same hospital in Guangdong province, the number of SNPs were less than 34, and the SNPs between XH1056 and XH1057 from Hebei province were 22, and the SNPs between XH1057 and XH1058 from Gansu province were 15, suggesting the existence of nosocomial transmission of these strains.The SNPs between the other strains were generally very far apart in the genome, indicating that the selection of the panel reaches a high level of heterogeneity, covering a wide diversity of strains (Fig. 1C).
The panel contains 31 known different Oxford STs as well as two strains (XH1056 and XH1057) that could not be classified as corresponding STs due to the lack of the Oxford scheme allele gdhB.According to the Pasteur scheme, the panel identified total 12 STs, including the most globally prevalent clone ST2, which is also overwhelmingly dominant in this panel (28/45), followed by ST40 (3/45) and ST215 (3/45).The panel contained 18 different capsule types known to be involved in capsule synthesis, with a further seven strains belonging to the novel KL type (7/45), which dominated the panel considerably, followed by KL34 (6/45), KL8 (5/45), and KL2 (4/45).Subsequent analysis of OCL loci revealed that OCL1c and OCL1d had the highest prevalence, accounting for 66.7% (30/45) of the whole panel isolates, followed by OCL5 (6/45) and OCL2 (2/45).Prediction of plasmid replicons (0-3 per strain) for this panel using the pAci database showed that AbGRI3-repAciN was most abundant, which was identified in 25 strains, while GR25 was found in 18 strains and GR24 in 15 strains (Fig. 2).

AST results and antibiotic resistance genes (ARGs)
The AST results showed that three isolates (XH1022, XH1832, and XH1034) were susceptible to all 10 antibiotics tested, and one isolate (XH1025) was non-susceptible to all tested antibiotics, except colistin.Specifically, 42 strains were resistant to car bapenems (imipenem and meropenem), and the minimum inhibitory concentrations were mainly in the range of 32-64 mg/L.Additionally, 36 strains were found to be resistant to aminoglycosides (amikacin and gentamicin), with 32 strains and 34 strains having extremely high MICs (MIC ≥256 mg/L) for amikacin and gentamicin, respectively; whereas 42 strains were identified as resistant to cephalosporins (ceftazidime and cefepime), and 39 strains were resistant to fluoroquinolones (ciprofloxacin).In contrast, resistance to tigecycline was very low (2/45, 4.4%), and only four strains were resistant to the new antimicrobial drug eravacycline.All strains in this panel were colistin-susceptible (Fig. 3A through J).
Overall, a variety of known ARGs were detected in this group of isolates, and a total of 65 different ARGs were found to be resistant to the six classes of antimi crobials (aminoglycosides, carbapenems, fluoroquinolones, tetracyclines, lipopeptides, and cephalosporins).All strains carried ant (3'')-IIa, bla ADC and bla OXA-51-like genes as expected, which are inherent to A. baumannii.The bla OXA-51-like family detected bla OXA-51 , bla OXA-66 , bla OXA-68 , bla OXA-69 , bla OXA-83 , bla OXA-132 , bla OXA-259 , bla OXA-260 , and bla OXA-430 , which also illustrates the significant variability among the strains in our collection.Of the 42 strains, carbapenemase genes were encoded by bla OXA-23 (n = 40), bla OXA-24 (n = 1), bla OXA-58 (n = 1), and bla NDM-1 (n = 1), with bla OXA-23 being the most common.One strain, XH1041, possessed two carbapenemase genes, bla OXA-23 and bla OXA-58 .In addition to carbapenem resistance genes, strains also carried aminoglycoside resistance genes such as aadA1, aph (3')-VIa, aph (3')-Ia, and aac (6')-Ib'.In the diversity panel of this study, a total of 39 strains were found to be resistant to ciprofloxacin.All of these resistant strains exhibited amino acid alterations in either the DNA gyrase (GyrA) or topoisomerase IV (ParC).The most common mutation observed was a combination of mutations in both genes, specifically GyrA (S81L) and ParC (S84L), which has been confirmed by previous research as the primary cause of fluoroquinolone resistance in A. baumannii (34,35).Furthermore, among these cephalosporin-resistant strains, 29 strains were found to have ISAba1 directly inserted upstream of the bla ADC gene, a mechanism that has been confirmed in earlier research as conferring resistance (36). A. baumannii XH1021 and XH1047 are resistant to tetracycline-class third-genera tion derivative tigecycline.Genomic analysis detected the insertion of ISAba1 within the adeN gene of XH1037, which has been proven to confer tigecycline resistance (26).The mechanism of tigecycline resistance in XH1021, however, remains to be fully elucidated.In 31 strains, we detected the tetracycline resistance gene tet(B), while 37 strains contained the sulfonamide resistance genes sul1 and sul2 (details in Fig. 2).

Comparison of resistance rates of isolates in ST2 vs non-ST2 and MTE vs non-MTE groups
The 45 strains of this panel were identified by MLST, and a total of 28 strains (62.2%, 28/45) belonged to ST2 (Pasteur scheme) isolates, which showed significantly higher resistance to cefepime, ceftazidime, imipenem, meropenem, ciprofloxacin, amikacin, and gentamicin than the non-ST2 group, whereas there was no difference in the resistance to colistin, tigecycline, and eravacycline among the isolates (Table 2).The morphology of the colonies on sheep blood plates also demonstrated the diversity of the panel.The panel strains were classified into three different types of groups according to previous descriptions (11), of which seven were MTA with an "oil on water" phenotype, four were MTC with a "raised center and irregular base" phenotype, and 34 were MTE with a "flat center and wavy edges" phenotype.In order to allow for intergroup comparisons, we chose to compare the MTE group with the non-MTE group.The MTE isolates differed only in their resistance to eravacycline, with none of the strains being resistant to eravacycline (P = 0.002), and there was no difference in resistance to any of the other antibiotics tested between the two groups (Table 3).a "ND"-indicates that no relevant mobile element was detected.
b "/"-indicates the presence of only a single-copy resistance gene, which is not statistically significant.

Heterogeneity of biofilm formation and growth rates
The assay data showed that all the strains had the ability to form biofilms, but each strain differed in its biofilm-forming ability, and heterogeneity was observed.Of particular interest is that XH1026 and XH1032 produced biofilms at a level comparable to that of the reference strain ATCC19606.ST2 isolates produced low biofilms, whereas MTE-type isolates produced strong biofilms (Fig. 8).In addition, the growth rates of all strains were highly variable, with rates in MHB (OD 600 ) ranging from 0.67 to 1.68.XH1018 showed minimal cost of fitness, i.e., a very rapid growth rate in the absence of antibiotic stress, whereas the relative growth rates of XH1026, XH1056, and XH1057 were very low.There was a significant difference in growth rates between isolates of ST2 and non-ST2, and there was no significant difference between groups with different colonial morphologies (Fig. 9).

Virulence phenotypes and relationship to specific KL types
Next, we performed in vivo virulence assays in Galleria mellonella on all different KL isolates.All tested A. baumannii strains exhibited less virulence than the positive virulence control AB5075, and within this, XH1024 and XH1047 were more virulent (Fig. 10A).To test the hypothesis if the capsule type correlates with virulence, we tested the virulence of three KL10 strains, XH1024, XH1029, and XH1031.The results showed that the virulence of XH1029 and XH1031 was very low when compared to that of XH1024, illustrating the heterogeneity among strains, even in strains of the same KL type (Fig. 10B).

DISCUSSION
A. baumannii is a common clinical pathogen that is easily transmitted in hospitals, causing a variety of serious infections due to its persistence in the environment and acquired multidrug resistance (38).In recent years, the rate of A. baumannii-associated infections, antibiotic resistance, and morbidity has been increasing, placing a serious burden on the global healthcare system (39).In order to provide protection against A. baumannii infections, researchers are actively exploring vaccines, antimicrobial peptides, phage therapeutics, or novel antimicrobial drugs (40)(41)(42)(43)(44).However, at this stage, the test strains used to evaluate these therapeutic candidates vary from study to study, coming from different sources and in varying numbers (often very large and with very little heterogeneity), which undoubtedly complicates the complexity of the studies, and more importantly, makes it difficult to establish the general effectiveness of the new treat ment/diagnostic.In addition, there are a considerable number of studies on A. baumannii pathogenesis and drug therapy that use reference strains only.However, it has been shown that these long-established and widely used "reference, " namely, ATCC19606 (ST52/KL3), ATCC17978 (ST437/KL3), and AB5075 (ST1/KL25), belong to rare sequence types.Their colony morphology and capsule formation are also limited compared to many other A. baumannii isolates (11).Thus, they cannot represent current clinical strains in terms of their phenotypic and genotypic diversity.Therefore, the research community would benefit from a standardized, heterogeneous panel of strains.
To the best of our knowledge, there were three A. baumannii panel repositories.The first one is a panel of 41 strains published jointly by the Centers for Disease Control and Prevention and Food and Drug Administration (CDC-FDA) (45); the second one is a panel of Acinetobacter spp.containing eight A. baumannii strains collected from a hospital in Seoul in South Korea (46); and the third one is a panel of 100 A. baumannii strains developed by the Multidrug-Resistant Organism Repository and Surveillance Network (MRSN) of the Walter Reed Army Research Institute in the United States (47).However, all of them have some shortcomings to a greater or lesser extent: all of them lack the phenotypic results of the strains.Although all the strains inclu ded in this panel were collected in China, it has representatives of the globally distrib uted clones, ST208 OXF , ST191 OXF , ST368 OXF, and ST369 OXF (48).These strains possess prevalent acquired resistance genes such as bla OXA-23 , bla OXA-24 , and bla OXA-58 , as well as the intrinsic OXA-51-like carbapenemases bla OXA-66 and bla OXA-69 (49).Furthermore, there are emerging sequence types, including ST452 OXF , ST684 OXF , and ST373 OXF (50,51).Previous studies established that KL2, KL10, KL22, and KL52 were the major types among the carbapenemase-resistant A. baumannii (CRAB), while also leading to more severe infections (especially pneumonia) and higher mortality (52).Several studies also reported KL49 to be a phenotype with higher virulence and clinical mortality (53).All of these capsular types were included in this study, which also provides a good resource for conducting capsular-related studies.Furthermore, a strain which harbors a novel resistance island AbGRI5 is the isolate XH1056, found in our panel (54).One major advantage of our panel is that we characterized the multiple phenotypes of the strains, allowing informed selection of strains based on specific biological questions.
ST2 A. baumannii is prevalent worldwide with multiple resistance genes and resistance plasmids, leading to wide dissemination of resistance; thus, ST2 is strongly correlated with resistance to a variety of antibiotics, such as cephalosporins, carbape nems, quinolones, and aminoglycosides (55).In addition to antimicrobial resistance, ST2 isolates commonly exhibit strong biofilm-forming capacity, which can increase their pathogenicity (56).In our study, ST2 was the most common sequence type, and ST2 isolates showed significantly higher rates of resistance to cefepime, ceftazidime, imipenem, meropenem, ciprofloxacin, amikacin, and gentamicin, while also not showing a strong loss of fitness, i.e., a lower cost of adaptation.Unsurprisingly, A. baumannii ST2 can be clinically predominant.Surprisingly, the ST2 strains in our panel produced lower  biofilms than those in the non-ST2 group (57).However, by comparing the laboratory characteristics of IC2 and non-IC2 A. baumannii isolates, the biofilm-forming ability of IC2 was found to be significantly lower than that of non-IC2 isolates (58), which is similar to our findings.Szczypta et al. collected all strains from two clonal outbreaks in the same hospital and showed that all ST2 strains tested had lower biofilm formation capacity than ATCC19606, which belongs to ST52 (59).Interestingly, previous studies observed that many multidrug resistant clinical isolates did not form biofilms.In contrast, a large number of strong biofilm producers were drug-sensitive A. baumannii strains; the biofilm can protect the bacteria by forming a barrier, preventing many antibiotics to reach the cells embedded within the biofilm (60,61).Research published to date has yielded confusing perspectives on the relationship between biofilms and bacterial resistance to antibiotics, with some studies indicating a positive correlation, others a negative correlation, or no relationship at all (62-64).Studies have reported that strains from ICU patients and non-burn patients produce more biofilms; furthermore, bacteria from clinical environments produce more biofilms than those from environmental sources (61,65,66).In fact, the formation of biofilms by strains is a rather complex process that not only accounts for individual differences among different strains but also takes into consideration other factors such as incubation time, nutritional media, and staining time.
When examining the methodologies regarding biofilms, it is apparent that there are variations in experimental conditions, and there is no clear standard for the absorbance value that determines whether the strains studied form biofilms (61).Consequently, relying solely on biofilm formation to predict antimicrobial resistance is one-sided and lacks credibility.These findings suggest that when exploring the relationship between biofilm formation and antimicrobial resistance, it is important to consider the source of the strain and the influence of external environments, as these may affect the final conclusions.Previous studies have observed the presence of genes such as bla PER-1 , bla TEM , tetB, bla oxa , and class 1 integrons in biofilm-producing strains (67,68), which may provide an alternative explanation for the relationship between biofilms and antimicrobial resistance, and further studies are needed to elucidate the mechanisms involved in this process.Given the spread of A. baumannii ST2 strains, especially in clinical settings, it is important to monitor the prevalence also to establish effective measures to limit the distribution of such strains.It has been shown that hypervirulent K. pneumoniae (hvKp) tends to exhibit a hypermucoviscous phenotype.In addition, studies have reported the emergence and spread of carbapenem-resistant K. pneumoniae (CRKP) with a novel "red, dry, and rough" morphology (rdar) (69).In the case of A. baumannii, multi-antibiotic resistance has also been reported for the mucoid phenotype (70).These studies suggest a relationship between colony morphology and bacterial antibiotic resistance and virulence.Differences in colony morphology may also potentially reflect differences in antibiotic resistance and virulence of strains.We divided the strains in this panel into three groups, as described in the literature (11), and further investigated the relationships between the MTE "flat center and wavy edge" phenotype with antibiotic resistance, biofilm, and growth rates.We found that strains with the MTE phenotype were more resistant to eravacycline and produced less biofilms, while there was no significant difference in their growth rates.However, due to the limited number of strains, the relationship between MTE phenotypes and bacterial antibiotic resistance, as well as biofilm production, needs to be investigated more thoroughly by increasing the number of samples.
The virulence phenotypes of the strains tested in this study also exhibit diversity, ranging from low to high virulence.In the future, these strains can be employed for virulence research, expanding beyond the use of just strain AB5075.Our data also indicate that there is no clear correlation between the KL type of A. baumannii and the strain's virulence.The virulence of A. baumannii is not only determined by a single factor but by many (71), which makes it impossible to link pathogenicity to a specific KL type.
One remaining issue with our panel is that our study does not include pan-resistant strains and also no colistin-resistant strains.Being limited in the number of isolates, our panel can of course not cover all of the clonal lineages, which is the case in any panel that exists to date.Thus, with the large number of strains globally, the constant emergence of novel clones, and the constant evolution of strains, it is impossible to completely cover all clones.Nonetheless, our research can be beneficial for the development of vaccines or for the isolation of novel phages for phage therapy (72,73).Nonetheless, this panel covers the major and most of the currently clinically problematic A. baumannii clones.
In reality, one significant factor contributing to the circulation of vari ous variants of reference strains among laboratories is improper handling of these strains.The ATCC clearly stipulates that strain passages should not exceed five generations (https://www.atcc.org/resources/technical-documents/reference-strains-how-many-passages-are-too-many).However, most researchers are not fully cognizant of it and often distribute strains to other laboratories without proper consideration.Researchers who receive these strains through such exchanges continue to distribute them indiscriminately to other colleagues.Over time, these strains undergo numerous passages, leading to genomic rearrangements.Consequently, it is plausible that different laboratories end up with multiple variants of the reference strains.To address this issue, we recommend that all individuals using strains from this panel take steps to prevent multiple passages, thereby preventing the appearance of more variants.It is crucial to adhere to proper strain management practices to maintain the integrity and reliability of the reference strains for accurate scientific research and testing.We believe that our panel of 45 different strains of A. baumannii from a collection of 2,197 A. baumannii will facilitate multiple medical, scientific, and translational studies to combat infections caused by this critical pathogen.

FIG 1 (
FIG 1 (A) Geographic distribution of clinical A. baumannii isolates collected in this study (beige area).The origin of the final strains included in the panel is indicated by filled black triangles.The source of this map is the Gaode Open Platform; (B) Phylogenetic tree of the panel strains with major human ICs.The ICs are shown in different colors, and the rings in the outer circle show the panel isolate groups; (C) Heatmap of 45 A. baumannii strains based on single-nucleotide polymorphisms (SNPs), and the distances of the SNPs between different strains are shown in the heatmap.

FIG 9
FIG 9 Growth rates.(A) Growth rates of panel strains; (B) comparison of ST2 and non-ST2 growth rates; (C) comparison of MTE and non-MTE growth rates.Data were analyzed by independent samples t-test and expressed as mean ± standard deviation, with asterisks indicating significant differences between groups (*P < 0.05, **P < 0.01, **P < 0.001, and ****P < 0.0001).

TABLE 1
Isolates carrying acquired carbapenem resistance genes, their localization, copy number, MIC of meropenem and imipenem, and their gene environment and structure

TABLE 2
Comparison of ST2 and non-ST2 resistance rates in A. baumannii

TABLE 3
Comparison of MTE and non-MTE resistance rates in A. baumannii